Halofuginone-guided nano-local therapy: Nano-thermosensitive hydrogels for postoperative metastatic canine mammary carcinoma with scar removal

Abstract

In female dogs, the highest morbidity and mortality rates cancer are the result of mammary adenocarcinoma, which presents with metastases in the lung. Other than early surgical removal, however, no special methods are available to treat mammary adenocarcinoma. Because human breast cancer and canine mammary carcinoma share clinical characteristics and heterogeneity, the canine model is a suitable spontaneous tumor model for breast cancer in humans. In this study, the physical swelling method was used to prepare halofuginone-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles nano-thermosensitive hydrogels (HTPM-gel). Furthermore, HTPM-gel was investigated via characterization, morphology, properties such as swelling experiment and in vitro release with reflecting its splendid nature. Moreover, HTPM-gel was further examined its capability to anti-proliferation, anti-migration, and anti-invasion. Ultimately, HTPM-gel was investigated for its in vivo anticancer activity in the post-operative metastatic and angiogenic canine mammary carcinoma. HTPM-gel presented spherical under transmission electron microscope (TEM) and represented grid structure under scanning electron microscope (SEM), with hydrodynamic diameter (HD) of 20.25 ± 2.5 nm and zeta potential (ZP) of 15.10 ± 1.82 mV. Additionally, HTPM-gel own excellent properties comprised of pH-dependent swelling behavior, sustained release behavior. To impede the migration, invasion, and proliferation of CMT-U27 cells, we tested the efficacy of HTPM-gel. Evaluation of in vivo anti-tumor efficacy demonstrates HTPM-gel exhibit a splendid anti-metastasis and anti-angiogenic ability, with exhibiting ideal biocompatibility. Notably, HTPM-gel also inhibited the scar formation in the healing process after surgery. In summary, HTPM-gel exhibited anti-metastasis and anti-angiogenic and scar repair features. According to the results of this study, HTPM-gel has encouraging clinical potential to treat tumors with multifunctional hydrogel.

Keywords: Angiogenesis; Halofuginone; Metastasis; Postoperative; Scar removal; Thermosensitive hydrogels.

Graphical abstract

Fig. 1

Characterization and morphology of the HTPM-gel. (A) The HTPM-gel was prepared via a physical swelling method. (B) Physical morphology of the HTPM-gel showed the solution status at 4 °C and the gel status at 37 °C after 1.5 min, which indicated the temperature sensitivity of the HTPM-gel. (C) We used transmission electron microscopy to observe the surface morphology of the HTPM-gel. (D) We used scanning electron microscopy to observe the surface morphology of the HTPM-gel. (E) (Differential Scanning Calorimetry) DSC analysis of HTPM-gel in situ gel depot samples were investigated via differential scanning calorimetry, n = 3. (F) Thermogravimetric analysis (TGA) of HTPM-gel in situ gel depot samples were determined by a thermogravimetric analyzer, n = 3. (G) The UV absorption spectrum indicated that the HTPM-gel had the largest characteristic absorption peak at 243 nm (n = 3).

Fig. 2

Properties of the HTPM-gel. (A) Swelling rate and (B) de-swelling rate of HTPM-gel were investigated at various time via determination of weight change in dissolved hydrogels, n = 3. (C) Swelling rate of HTPM-gel were explored by different pH conditions, n = 3. (D) Using the dialysis bag method, we investigated the in vitro drug release HF profiles of the HTPM and HTPM-gel in a PBS solution (pH 7.2–7.4) (n = 3). (E) We used 27G, 29G, and 30G needles to measure the maximal injection force for HTPM-gel (n = 3). (F) We used a 29G needle to measure variations in injection force as a function of HTPM-gel concentration (n = 3).

Fig. 3

The impact of the HTPM-gel on migration, clone formation, invasion activity for CMT-U27 cells. The HTPM-gel significantly inhibited migration (A&B), (C&D) clone formation, and (E&F) invasion of CMT-U27 cells compared with PBS and Blank-gel (n = 3).

Fig. 4

HTPM-gel administrated in situ had anticancer effects against postoperative orthotopic CMC xenografts in nude mice. (A) Pattern diagram, (B) body weights, (C) tumor volumes, (D) morphology of the harvested tumors, (E) tumor weights, and (F) TUNEL staining analysis (400×) of the degree of tumor necrosis and apoptosis rates (G) for drug-untreated nude mice or after administration of the Blank-gel, HTPM-gel (n = 5). The tumor images of the HTPM-gel are shown in black arrows, which indicate apoptosis of the tumor tissue induced from HTPM-gel. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

HTPM-gel suppressed lung metastasis. Histological analysis using H&E staining of lungs from orthotopic tumor-bearing mice treated with the Blank-gel, HTPM-gel (200×). The black solid arrow and black box area in the lung slices of the Untreated and Blank-gel groups indicate lungs that appeared to be or were filled with canine mammary carcinoma parenchyma cells. Lung metastasis of tumor tissues from orthotopic CMC tumor-bearing mice treated with the Blank-gel, HTPM-gel. The black-dotted arrow in the analysis of STC-1 and BRCA2 in the Untreated and Blank-gel groups indicate inhibition of lung metastasis of the tumor tissue (400×).

Fig. 6

Prognosis of mice with orthotopic tumors treated with the Blank-gel, HTPM-gel. (A) NLR and (B) LMR B) (NLR: neutrophil granulocyte/lymphocyte ratio, LMR: lymphocyte/monocyte ratio) of nude mice after administration of Untreated, Blank gel, HTPM-gel (n = 5). (C&D) We used an ELISA regent kit to detect the representative inflammatory factors secreted by Th cells, such as IL-22 and IL-17, from the serum (n = 5). (E) Histological analysis of spleens from mice with orthotopic tumors treated with the Blank-gel, HTPM-gel using H&E staining (200×). The black open arrows, black solid arrows, black dotted arrows, and black square-pointed arrows in the spleen images represent the neutrophils, infiltrating lymphocytes, monocytes, and macrophages, respectively, of the Untreated and Blank-gel groups.

Fig. 7

Effect of HTPM-gel on the scar from the postoperative site. (A) Representative images indicated the excellent scar removal capability of HTPM-gel which was shown in the recovery period of CMC bearing mice. (B) Collagen I and TGF-β, as key factors in scar formation, were investigated via immunochemical technique (400×). Black dovetail arrow in the Collagen I image of the Untreated and Blank gel group indicate scar formation of the skin tissue. Black open arrow in the TGF-β images of the HTPM-gel indicates healing degree of the skin tissue. (C&D) We used an ELISA regent kit to detect representative inflammatory factors, such as IL-6 and TNF-α, from the serum (n = 5).

Fig. 8

Safety evaluation of biocompatibility of orthotopic CMC-bearing mice treated with the Blank-gel, HTPM-gel. (A) Representative images of primary organs such as heart, liver and kidney in mice from H&E staining (200×), n = 5. (B&C) Representative blood physiology indicators such as hemoglobin (HGB) and platelet (PLT) in the serum of mice were determined, n = 5. (D&E) We determined alanine transaminase (ALT) and serum aspartate transaminase (AST) as the representative blood biochemical indicators of mice (n = 5).